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Schematic overview of the early secretory pathway in mammalian cells. Abbreviations: ER, endoplasmic reticulum; ERGIC, endoplasmic reticulum-Golgi intermediate compartment; COG complex, conserved oligomeric Golgi complex; COPI, coat protein complex I.
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Glycosylation is an important post-translational modification for both intracellular and secreted proteins. For glycosylation to occur, cargo must be transported after synthesis through the different compartments of the Golgi apparatus where distinct monosaccharides are sequentially bound and trimmed, resulting in increasingly complex branched glyc...
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... models for the trafficking routes in the Golgi exist, but the most favorable model of membrane traffic within the Golgi is the cisternal maturation model (Figure 1) [56]. Cisternal maturation is the gradual conversion of a Golgi compartment by the delivery of proteins and lipids from more mature Golgi compartments concomitant with the removal of Golgi proteins and lipids from previous Golgi compartments by coatomer (coat protein complex I; COPI)-mediated retrograde membrane trafficking [27,56]. ...
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The Conserved Oligomeric Golgi (COG) complex, a multi-subunit vesicle tethering complex of the CATCHR (Complexes Associated with Tethering Containing Helical Rods) family, controls several aspects of cellular homeostasis by orchestrating retrograde vesicle traffic within the Golgi. The COG complex interacts with all key players regulating intra-Gol...
Citations
... Thus, tight regulation of trafficking between the ER and Golgi compartments is crucial on the one hand, for the processing and PTMs of proteins from the secretory pathway, and on the other hand, for the proper localization of ER-and Golgi-resident proteins [63][64][65][66]. Indeed, defects in intra-Golgi transport of enzymes implicated in glycosylation strongly affect the maturation, processing and trafficking of glycoconjugates, leading to severe pathologies such as congenital disorders of glycosylation (CDG) [67]. The early steps of the canonical protein secretion pathway are governed by COPII and COPI-mediated transports [1][2][3] (Figure 2b,c), that are both sensitive to O-GlcNAc homeostasis. ...
The transport of proteins between the different cellular compartments and the cell surface is governed by the secretory pathway. Alternatively, unconventional secretion pathways have been described in mammalian cells, especially through multivesicular bodies and exosomes. These highly sophisticated biological processes rely on a wide variety of signaling and regulatory proteins that act sequentially and in a well-orchestrated manner to ensure the proper delivery of cargoes to their final destination. By modifying numerous proteins involved in the regulation of vesicular trafficking, post-translational modifications (PTMs) participate in the tight regulation of cargo transport in response to extracellular stimuli such as nutrient availability and stress. Among the PTMs, O-GlcNAcylation is the reversible addition of a single N-acetylglucosamine monosaccharide (GlcNAc) on serine or threonine residues of cytosolic, nuclear, and mitochondrial proteins. O-GlcNAc cycling is mediated by a single couple of enzymes: the O-GlcNAc transferase (OGT) which catalyzes the addition of O-GlcNAc onto proteins, and the O-GlcNAcase (OGA) which hydrolyses it. Here, we review the current knowledge on the emerging role of O-GlcNAc modification in the regulation of protein trafficking in mammalian cells, in classical and unconventional secretory pathways.
... Transmembrane protein 199 (TMEM199) is an assembly factor of the V-ATPase required for the acidification of the endosomes of the secretory pathway, including the lysosome and the Golgi apparatus. Insufficient Golgi acidification may hamper Golgi glycosylation machinery leading to a glycosylation defect (Linders et al., 2020). In 2016 Jansen et al., identified different TMEM199 pathogenic variants in four previously unsolved cases of CDG (Calvo et al., 2008) and Vajro et al. reported three additional patients with a similar phenotype in 2018. ...
Congenital disorders of glycosylation (CDG) are genetic multisystem diseases, characterized by defective glycoconjugate synthesis. A small number of CDG with isolated liver damage have been described, such as TMEM199-CDG, a non-encephalopathic liver disorder with Wilson disease-like phenotype. Only eight patients with TMEM199-CDG have been described including seven Europeans (originating from Greece and Italy) and one Chinese. Three patients from southern Italy (Campania) shared the same known missense mutation pathogenetic variant NM_152464.3:c. 92G > C (p.Arg31Pro), also found in a Greek patient. Here we report a new patient from southern Italy (Sicily), with a homozygous c.92G > C p.(Arg31Pro) variant in TMEM199. The patient's phenotype is characterized by mild non-progressive hepatopathy with a normal hepatic echo structure. A persistent increase in serum transaminases, total and low-density lipoprotein cholesterol and low serum ceruloplasmin and copper levels and normal urinary copper excretion were observed. Matrix-assisted laser desorption/ionization mass spectrometry analyses showed abnormal N- and O- protein glycosylation, indicative of a Golgi processing defect and supporting the function of TMEM199 in maintaining Golgi homeostasis. TMEM199-CDG is an ultra-rare CDG relatively frequent in the southern Mediterranean area (7 in 9 patients, 77%). It is mainly associated with the c.92G > C (p.Arg31Pro) pathogenetic allele globally reported in 4 out of 7 families (57%), including one from Greece and three unrelated families from southern Italy. The almost uniform clinical phenotype described in patients with TMEM199-CDG appears to reflect a higher prevalence of the same variant in patients from the southern Mediterranean area.
... The GC is involved in protein quality control and is the fundamental organelle for the glycosylation of numerous proteins. To this aim, the GC is a highly dynamic organelle, organized in flattened polarized cisternae, organized in a cis region facing the ER and trans region directed toward the plasma membrane [8,24,25]. Transport between the ER and the GC is regulated by the COP II and COP I coat vesicles. ...
... In fact, an alteration of its morphology potentially leads to damages in the organization of the sub-compartments and can introduce an impairment of the post-translation processing as a consequence. In turn, the accumulation of improperly modified proteins affects GC organization, vesicles assembly, and protein traffic itself [24,27]. Altogether, this evidence confers a central role to the GC [9]. ...
A growing number of disorders has been associated with mutations in the components of the vesicular transport machinery. The early secretory pathway consists of Endoplasmic Reticulum, numerous vesicles, and the Golgi Complex (GC), which work together to modify and package proteins to deliver them to their destination. The GC is a hub organelle, crucial for organization of the other secretory pathway components. As a consequence, GC’s form and function are key players in the pathogenesis of several disorders. Skeletal muscle (SKM) damage can be caused by defective protein modifications and traffic, as observed in some Limb girdle muscular dystrophies. Interestingly, in turn, muscle damage in Duchenne dystrophic SKM cells also includes the alteration of GC morphology. Based on the correlation between GC’s form and function described in non-muscle diseases, we suggest a key role for this hub organelle also in the onset and progression of some SKM disorders. An altered GC could affect the secretory pathway via primary (e.g., mutation of a glycosylation enzyme), or secondary mechanisms (e.g., GC mis-localization in Duchenne muscles), which converge in SKM cell failure. This evidence induces considering the secretory pathway as a potential therapeutic target in the treatment of muscular dystrophies.
... As ATP6V1A is widely expressed, the observed deviation from physiological glycosylation could derive from either reduced activity of pH-dependent enzymes, such as sialyltransferases and fucosyltransferase, and/or from their mislocalization, due to non-optimal acidification of Golgi cisternae, or both. 26 An additional downstream impact in Golgi homeostasis by the altered lysosomal acidification is possible, as also hypothesized for ATP6AP2 deficiency. 27 Altered antennary fucosylation of plasma/serum glycoprotein has been documented in different diseases, [28][29][30] including developmental disorders. ...
Vacuolar-type H+-ATPase (V-ATPase) is a multimeric complex present in a variety of cellular membranes that acts as an ATP-dependent proton pump and plays a key role in pH homeostasis and intracellular signalling pathways. In humans, 22 autosomal genes encode for a redundant set of subunits allowing the composition of diverse V-ATPase complexes with specific properties and expression. Sixteen subunits have been linked to human disease.
Here we describe 26 patients harbouring 20 distinct pathogenic de novo missense ATP6V1A variants, mainly clustering within the ATP synthase α/β family-nucleotide-binding domain. At a mean age of 7 years (extremes: 6 weeks, youngest deceased patient to 22 years, oldest patient) clinical pictures included early lethal encephalopathies with rapidly progressive massive brain atrophy, severe developmental epileptic encephalopathies and static intellectual disability with epilepsy. The first clinical manifestation was early hypotonia, in 70%; 81% developed epilepsy, manifested as developmental epileptic encephalopathies in 58% of the cohort and with infantile spasms in 62%; 63% of developmental epileptic encephalopathies failed to achieve any developmental, communicative or motor skills. Less severe outcomes were observed in 23% of patients who, at a mean age of 10 years and 6 months, exhibited moderate intellectual disability, with independent walking and variable epilepsy. None of the patients developed communicative language. Microcephaly (38%) and amelogenesis imperfecta/enamel dysplasia (42%) were additional clinical features. Brain MRI demonstrated hypomyelination and generalized atrophy in 68%. Atrophy was progressive in all eight individuals undergoing repeated MRIs.
Fibroblasts of two patients with developmental epileptic encephalopathies showed decreased LAMP1 expression, Lysotracker staining and increased organelle pH, consistent with lysosomal impairment and loss of V-ATPase function. Fibroblasts of two patients with milder disease, exhibited a different phenotype with increased Lysotracker staining, decreased organelle pH and no significant modification in LAMP1 expression. Quantification of substrates for lysosomal enzymes in cellular extracts from four patients revealed discrete accumulation. Transmission electron microscopy of fibroblasts of four patients with variable severity and of induced pluripotent stem cell-derived neurons from two patients with developmental epileptic encephalopathies showed electron-dense inclusions, lipid droplets, osmiophilic material and lamellated membrane structures resembling phospholipids. Quantitative assessment in induced pluripotent stem cell-derived neurons identified significantly smaller lysosomes.
ATP6V1A-related encephalopathy represents a new paradigm among lysosomal disorders. It results from a dysfunctional endo-lysosomal membrane protein causing altered pH homeostasis. Its pathophysiology implies intracellular accumulation of substrates whose composition remains unclear, and a combination of developmental brain abnormalities and neurodegenerative changes established during prenatal and early postanal development, whose severity is variably determined by specific pathogenic variants.
... In agreement with previously published data, we found that GS15 and GS28 are actively incorporated into CCD vesicles to operate as v-SNARE proteins, while STX5 and Ykt6 remain on the Golgi and work as t-SNAREs (Zhang and Hong, 2001). STX5 is a transmembrane protein, which is shown to cycle via ER (Rowe et al., 1998;Bentley et al., 2006;Cottam and Ungar, 2012;Linders et al., 2020); it will be important to investigate which membrane carriers are used to recycle STX5 during Golgi biogenesis (Rowe et al., 1998). Curiously, previous proteomic studies identified STX5 as a component of in vitro formed COPI Golgi vesicles (Cottam and Ungar, 2012;Adolf et al., 2019;Linders et al., 2019) this result is likely to indicate the principal difference between in vivo accumulated and in vitro formed Golgi-derived vesicles. ...
Conserved Oligomeric Golgi (COG) complex controls Golgi trafficking and glycosylation, but the precise COG mechanism is unknown. The auxin-inducible acute degradation system was employed to investigate initial defects resulting from COG dysfunction. We found that acute COG inactivation caused a massive accumulation of COG-dependent (CCD) vesicles that carry the bulk of Golgi enzymes and resident proteins. v-SNAREs (GS15, GS28) and v-tethers (giantin, golgin84, and TMF1) were relocalized into CCD vesicles, while t-SNAREs (STX5, YKT6), t-tethers (GM130, p115), and most of Rab proteins remained Golgi-associated. Airyscan microscopy and velocity gradient analysis revealed that different Golgi residents are segregated into different populations of CCD vesicles. Acute COG depletion significantly affected three Golgi-based vesicular coats- COPI, AP1, and GGA, suggesting that COG uniquely orchestrates tethering of multiple types of intra-Golgi CCD vesicles produced by different coat machinery. This study provided the first detailed view of primary cellular defects associated with COG dysfunction in human cells.
... Serum transferrin glycoform analysis is the first-line laboratory test for the diagnosis of Nglycosylation defects: an increase of asialylated and/or disialylated transferrin or increased mono-, di-tri-and/or asialotransferrin are observed in CDG-I and CDG-II, respectively. The analysis of mucin type O-glycosylated apolipoprotein CIII (apoCIII) is informative for the diagnosis of protein O-glycosylation as well as combined Nand O-glycosylation disorders (Linders, Peters, Ter Beest, Lefeber, & van den Bogaart, 2020). ...
... Number of patients a References hyposialylation as in other COG defects (Linders et al., 2020;Palmigiano et al., 2017). Further studies are needed to characterize the impact of COG subunit deficiency on the patho-mechanisms of birth defects observed in COG-CDG, particularly in COG6-CDG. ...
Background:
Deficiency of Conserved Oligomeric Golgi (COG) subunits (COG1-8) is characterized by both N- and O-protein glycosylation defects associated with destabilization and mislocalization of Golgi glycosylation machinery components (COG-CDG). Patients with COG defects present with neurological and multisystem involvement and possible malformation occurrence. Eighteen patients with COG6-CDG (COG6 mutations) were reported to date. We describe a patient with COG6-CDG with novel variants and a novel clinical feature namely a congenital recto-vaginal fistula.
Methods:
In-depth serum N- and O-glycosylation structural analyses were conducted by MALDI-TOF mass spectrometry. COG6 variants were identified by a gene panel and confirmed by Sanger sequencing.
Results:
This female newborn presented with facial dysmorphism, distal arthrogryposis and recurrent stool discharges per vaginam. A double-contrast barium-enema X-ray study revealed a dehiscence (approximately 5 mm) at the anterior wall of the rectal ampoule communicating with the vagina consistent with a recto-vaginal fistula. She had developmental delay, corpus callosum dysgenesis, liver and gastrointestinal involvement, hyperthermia episodes and early demise. Serum N- and O-glycosylation analyses pointed to a profound Golgi disarrangement. We identified two novel variants in COG6: a deletion of 1 bp mutation c.823delA creating a shift in the reading frame and a premature stop codon and a 3 bp deletion (c.1141_1143delCTC) producing an in-frame deletion of 1 amino acid.
Conclusion:
The congenital recto-vaginal fistula is a rare type of anorectal malformation that, to our knowledge, has not been reported in patients with a COG6 defect nor in patients with other COG defects. This study broadens COG6-CDG genetic landscape and spectrum of malformations.
... 1,2 pH is not only crucial for proper protein folding and enzyme activity through influencing the charge of amino acid side chains, but its importance in secretory protein transport is increasingly clear. 4 pH affects binding affinities of cargo molecules to trafficking chaperones and thereby pH differences facilitate intracellular transport by both influencing the transit of cargo 5−11 and the sorting of secretory pathway resident proteins. 12−14 Moreover, the localization of glycosylation enzymes and their substrates is determined by pH, 4,15−18 and defects in this homeostasis cause a wide range of human disease. ...
Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein, pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution is unsuitable to follow the rapid transit of cargo between organelles. Therefore, we applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway and compared the pH values obtained by the FLIM-based measurements with those obtained by conventional ratiometric imaging. Then, we analyzed the dynamic pH changes within cells treated with Bafilomycin A1, to block the vesicular ATPase, and Brefeldin A, to block endoplasmic reticulum (ER)–Golgi trafficking. Finally, we followed the pH changes of newly synthesized molecules of the inflammatory cytokine tumor necrosis factor-α while they were in transit from the ER via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution and can be used to assess organellar pH in disease models.
... The observed shift in size of VWF dimers could perhaps be explained by incomplete glycosylation of VWF as a result of mislocalized glycosylation enzymes that normally reside in the different Golgi stacks of the Golgi. 47 Therefore, we can hypothesize that an essential role of STX5 is to maintain a continuous Golgi from which properly multimerized VWF with post-translational modifications can be stored in elongated organelles, which have the capacity to mature into a stimulus-sensitive WPB. ...
Von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells (ECs). VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPBs), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde ER-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometrybased approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in ECs using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted ECs exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion-incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. Taken together, our study has identified SNARE protein STX5 as a novel regulator of WPB biogenesis.
... CDG screening revealed a strong hyposialylation of protein N-glycosylation and mucin-type O-glycosylation, as analyzed by isofocusing of, respectively, plasma transferrin ( Fig. 1a and Supplementary Table 2) and apolipoprotein CIII (ApoCIII-IEF, Fig. 1b and Supplementary Table 2). ApoCIII-IEF showed a strong increase of non-sialylated apoCIII (ApoCIII-0) band intensities compared to the intensities of the fully glycosylated form, even stronger than those observed for genetic defects in the conserved oligomeric Golgi (COG) complex, a known group of disorders with disturbed Golgi homeostasis and abnormal glycosylation 26 . ...
The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein syntaxin-5 (Stx5) is essential for Golgi transport. In humans, the STX5 mRNA encodes two protein isoforms, Stx5 Long (Stx5L) from the first starting methionine and Stx5 Short (Stx5S) from an alternative starting methionine at position 55. In this study, we identify a human disorder caused by a single missense substitution in the second starting methionine (p.M55V), resulting in complete loss of the short isoform. Patients suffer from an early fatal multisystem disease, including severe liver disease, skeletal abnormalities and abnormal glycosylation. Primary human dermal fibroblasts isolated from these patients show defective glycosylation, altered Golgi morphology as measured by electron microscopy, mislocalization of glycosyltransferases, and compromised ER-Golgi trafficking. Measurements of cognate binding SNAREs, based on biotin-synchronizable forms of Stx5 (the RUSH system) and Förster resonance energy transfer (FRET), revealed that the short isoform of Stx5 is essential for intra-Golgi transport. Alternative starting codons of Stx5 are thus linked to human disease, demonstrating that the site of translation initiation is an important new layer of regulating protein trafficking.
... V-ATPase is a proton pump that transports H+ via active transport and provides a homeostatic pH environment for various cellular activities [5]. For example, maintaining the pH gradient from the cis to the trans Golgi apparatus ensures proper protein post-translational modifications and targeting [3,6]. ...
Background:
The ATP6AP1 gene coding for the accessory protein Ac45 of the vacuolar-type adenosine triphosphatases (V-ATPase) is located on chromosome Xq28. Defects in certain subunits or accessory subunits of the V-ATPase can lead to congenital disorders of glycosylation (CDG). CDG is a group of metabolic disorders in which defective protein and lipid glycosylation processes affect multiple tissues and organs. Therefore, the clinical presentation of patients with ATP6AP1-CDG varies widely. In this report, we present a case of ATP6AP1-CDG in a Chinese infant, with clinical features and genotype.
Case summary:
An 8-mo-old boy was admitted to our hospital because unexplained hepatosplenomegaly and elevated transaminases that had been noted while he was being treated for a cough at a local hospital. A post-admission examination at our hospital revealed abnormalities in the infant's liver, brain, and immune system. Trio-based whole exome gene analysis identified a hemizygous pathogenic mutation c.1036G>A (p.E346K) in exon 9 of the ATP6AP1 gene. This variant of the ATP6AP1 gene has not been reported in East Asian countries until now.
Conclusion:
Based on the infant's clinical manifestations and the results of genetic detection, he was clearly diagnosed with ATP6AP1-CDG. The clinical manifestations of children with CDG vary widely. Genetic testing analysis helps in the clinical diagnosis of children with CDG.
